Quantitation of HIV-1 proviral DNA relative to cellular DNA by the polymerase chain reaction.
We developed a quantitative assay for human immunodeficiency virus type 1 (HIV-1) proviral DNA sequences using the polymerase chain reaction (PCR). The relative copy numbers of HIV-1 proviral DNA molecules were determined by coamplification of an HIV-1 gag sequence and a portion of the DQ alpha locus of the histocompatibility (HLA) region. Because of the disparity in the copy number of cellular and HIV-1 templates, an attenuation in the efficiency of the HLA amplification was required to achieve simultaneous amplification and quantitation of both target sequences. The HIV-1 and HLA amplified products were detected by hybridization with radioactively labeled probes and the amount of probe bound to each product was determined with a radioanalytic system. Standard curves were generated by plotting the HIV-1 and HLA signals made against known copies of each target present prior to amplification. The copies of HIV-1 target relative to the number of cells in a given sample were determined by interpolation from standard curves. The procedure described here is generally applicable to the quantitation of other retroviruses.
Kellogg
D E
DE
Department of Infection Diseases, Cetus Corporation, Emeryville, California 94608.
Sninsky
J J
JJ
Kwok
S
S
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D005784:Gene Amplification; D015750:Genes, gag / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006683:HLA-DQ Antigens / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics*; D013698:Templates, Genetic
D004279:DNA, Viral; D006683:HLA-DQ Antigens; D004247:DNA